The ability of blastomeres from an early embryo to acquire a distinct program of cell fate is fundamental to the development of a multicellular organism. The ultimate goal of this research is to understand how cell fate becomes specified by cell interactions during regulative embryogenesis. Specifically this proposal examines the mechanisms and origins of control during the ontogeny of the sea urchin endoderm lineage. This lineage is chosen for several reasons: 1) its specification and differentiation during embryogenesis is regulated by cell interactions, 2) genes responsive to the inductive interactions have recently been cloned, 3) embryology of the sea urchin is classically described and amenable to manipulation, and 4) endoderm differentiation is fundamental to gastrulation in establishing the basic body plan of the embryo. Three features of endoderm ontogeny will be examined: 1) Examine the regulation of endoderm differentiation. Presumptive endodern cells will be dissociated from the embryo and cultured in vitro to test the hypothesis that differentiation is regulated by exogenous signals. Molecules of the extracellular matrix and growth factors will be tested. Differentiation will be assayed by gene activity specific to endoderm cells. Results from these experiments will be tested in vivo and will be compared to blastomeres normally restricted to non-endodermal cell fates - but which can be induced to an endodermal fate. 2) Investigate the mechanism for endoderm specific gene expression. These experiments will examine where in the embryo the specific transcriptional machinery originates for an endoderm program. That is, are components of the transcriptional machinery maternally derived and then activated locally in presumptive endoderm blastomeres, or are they produced by restricted zygotic transcription. The hypothesis posed is that the nuclear factors involved in this lineage specific transcription are activated by early cell interactions. To test this hypothesis, LvNI.2, a gene expressed specifically by endoderm cells will be examined for cis- regulatory elements important for temporal and spatial transcriptional regulation. These experiments will utilize an embryonic gene transfer system coupled with lac Z and CAT reporter genes. Such cis-regions will be used to examine the ontogeny of functional trans-acting factors in normal embryogenesis, in response to the extracellular matrix, and in non-endodermal lineages induced to an endoderm fate. 3) Determine the origin of extracellular matrix molecules which influence endoderm differentiation. The regulated ontogeny of the extracellular matrix (ECM) will be examined using molecular cloning techniques. The hypothesis will be tested that the extracellular matrix derived from maternal stores is important in the stimulation of zygotic ECM synthesis. This study will include the characterization of several novel ECM molecules.